This application extends a "proof of principle" study towards developing improved vaccines to infectious disease in response to the threat of bioterrorism. Our proposal addresses the hypothesis that anti-CD40 mAb and Toll-like receptor (TLR) agonist treatments can enhance the immune response to vaccinia virus and cross-reactive epitopes derived from the Category A agent smallpox/variola major virus. We will employ as a vaccine strain, two isolates of vaccinia virus (VV): Western Reserve (WR) and modified Ankara (MVA). VV (WR) is a long-standing model for human smallpox, and is the basis for the highly effective, classic DryVax vaccine. MVA is a replication-negative strain, appealing due to its increased safety. A substantial literature shows anti-CD40 mAb treatment and TLR agonists can augment DC activation, CD8+ CTL expansion and function, and other antiviral responses. The question is: can these biological 'adjuvants' enhance protective immunity and memory to MVA/VV epitopes, including those shared with smallpox? With pilot-project funding from the New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases, we have already examined an extensive panel of TLR agonists and anti-CD40 mAb combinations. We have shown that MVA is much less immunogenic than VV. We also have evidence that the limiting factor in the reduced immunogenicity of MVA is not decreased viral antigen expression, but rather the lack of sufficient inflammatory stimulation. However, the response against MVA can be substantially improved by a single co-injection of anti-CD40 and/or TLR agonist to levels approximating that following WR-VV immunization. Here, we propose to refine these novel strategies in murine model systems to enhance the acute and memory CTL, IFN-y producing and antibody responses to antigens encoded by MVA, including predicted epitopes of variola major/smallpox. The specific aims are to test the most promising anti-CD40 mAb and/or TLR agonist(s) for their ability to: 1) enhance serum Ab and cell-mediated immune responses to MVA, and 2) provide in vivo protection from a WR-VV challenge. The unique treatment combination of anti-CD40 mAb and TLR agonists should substantially augment these antiviral responses and allow the use of lower doses of MVA, thus reducing toxicity. In this way we hope to define new vaccination paradigms that can be tested in non-human primate pox systems and ultimately in man. [unreadable] [unreadable] [unreadable]